A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3 receptor function. 2010

Dai-Fei Wu, and Nidaa A Othman, and Douglas Sharp, and Arjun Mahendra, and Tarek Z Deeb, and Tim G Hales
Department of Pharmacology & Physiology, George Washington University, Washington, DC 20037, USA.

The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT(3)) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the gamma-aminobutyric acid type A (GABA(A)) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT(3)A receptors, we replaced it with alanine and expressed the 5-HT(3)A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT(3)A(C312A) mutant, when co-expressed with the wild-type 5-HT(3)A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT(3)A(C312A) with 5-HT(3)B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT(3)A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor delta and gamma subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor alpha7 and GABA(A) subunits) supported robust function. Thus, 5-HT(3)A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011978 Receptors, Nicotinic One of the two major classes of cholinergic receptors. Nicotinic receptors were originally distinguished by their preference for NICOTINE over MUSCARINE. They are generally divided into muscle-type and neuronal-type (previously ganglionic) based on pharmacology, and subunit composition of the receptors. Nicotinic Acetylcholine Receptors,Nicotinic Receptors,Nicotinic Acetylcholine Receptor,Nicotinic Receptor,Acetylcholine Receptor, Nicotinic,Acetylcholine Receptors, Nicotinic,Receptor, Nicotinic,Receptor, Nicotinic Acetylcholine,Receptors, Nicotinic Acetylcholine
D003545 Cysteine A thiol-containing non-essential amino acid that is oxidized to form CYSTINE. Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D012701 Serotonin A biochemical messenger and regulator, synthesized from the essential amino acid L-TRYPTOPHAN. In humans it is found primarily in the central nervous system, gastrointestinal tract, and blood platelets. Serotonin mediates several important physiological functions including neurotransmission, gastrointestinal motility, hemostasis, and cardiovascular integrity. Multiple receptor families (RECEPTORS, SEROTONIN) explain the broad physiological actions and distribution of this biochemical mediator. 5-HT,5-Hydroxytryptamine,3-(2-Aminoethyl)-1H-indol-5-ol,Enteramine,Hippophaine,Hydroxytryptamine,5 Hydroxytryptamine
D017124 Conserved Sequence A sequence of amino acids in a polypeptide or of nucleotides in DNA or RNA that is similar across multiple species. A known set of conserved sequences is represented by a CONSENSUS SEQUENCE. AMINO ACID MOTIFS are often composed of conserved sequences. Conserved Sequences,Sequence, Conserved,Sequences, Conserved
D044406 Receptors, Serotonin, 5-HT3 A subclass of serotonin receptors that form cation channels and mediate signal transduction by depolarizing the cell membrane. The cation channels are formed from 5 receptor subunits. When stimulated the receptors allow the selective passage of SODIUM; POTASSIUM; and CALCIUM. Serotonin 3 Receptor,5-HT3 Receptor,5-Hydroxytryptamine-3 Receptor,Receptor, Serotonin 3,Receptor, Serotonin, 5-HT3 Subunit A,Receptor, Serotonin, 5-HT3 Subunit B,Receptor, Serotonin, 5-HT3 Subunit C,Receptor, Serotonin, 5-HT3 Subunit D,Receptor, Serotonin, 5-HT3 Subunit E,Receptor, Serotonin, 5-HT3A,Receptor, Serotonin, 5-HT3B,Receptor, Serotonin, 5-HT3C,Receptor, Serotonin, 5-HT3D,Receptor, Serotonin, 5-HT3E,Serotonin 3 Receptors,5 HT3 Receptor,5 Hydroxytryptamine 3 Receptor,Receptor, 5-Hydroxytryptamine-3,Receptors, Serotonin 3
D018408 Patch-Clamp Techniques An electrophysiologic technique for studying cells, cell membranes, and occasionally isolated organelles. All patch-clamp methods rely on a very high-resistance seal between a micropipette and a membrane; the seal is usually attained by gentle suction. The four most common variants include on-cell patch, inside-out patch, outside-out patch, and whole-cell clamp. Patch-clamp methods are commonly used to voltage clamp, that is control the voltage across the membrane and measure current flow, but current-clamp methods, in which the current is controlled and the voltage is measured, are also used. Patch Clamp Technique,Patch-Clamp Technic,Patch-Clamp Technique,Voltage-Clamp Technic,Voltage-Clamp Technique,Voltage-Clamp Techniques,Whole-Cell Recording,Patch-Clamp Technics,Voltage-Clamp Technics,Clamp Technique, Patch,Clamp Techniques, Patch,Patch Clamp Technic,Patch Clamp Technics,Patch Clamp Techniques,Recording, Whole-Cell,Recordings, Whole-Cell,Technic, Patch-Clamp,Technic, Voltage-Clamp,Technics, Patch-Clamp,Technics, Voltage-Clamp,Technique, Patch Clamp,Technique, Patch-Clamp,Technique, Voltage-Clamp,Techniques, Patch Clamp,Techniques, Patch-Clamp,Techniques, Voltage-Clamp,Voltage Clamp Technic,Voltage Clamp Technics,Voltage Clamp Technique,Voltage Clamp Techniques,Whole Cell Recording,Whole-Cell Recordings
D019943 Amino Acid Substitution The naturally occurring or experimentally induced replacement of one or more AMINO ACIDS in a protein with another. If a functionally equivalent amino acid is substituted, the protein may retain wild-type activity. Substitution may also diminish, enhance, or eliminate protein function. Experimentally induced substitution is often used to study enzyme activities and binding site properties. Amino Acid Substitutions,Substitution, Amino Acid,Substitutions, Amino Acid

Related Publications

Dai-Fei Wu, and Nidaa A Othman, and Douglas Sharp, and Arjun Mahendra, and Tarek Z Deeb, and Tim G Hales
August 2009, Journal of neurochemistry,
Dai-Fei Wu, and Nidaa A Othman, and Douglas Sharp, and Arjun Mahendra, and Tarek Z Deeb, and Tim G Hales
November 1995, DNA and cell biology,
Dai-Fei Wu, and Nidaa A Othman, and Douglas Sharp, and Arjun Mahendra, and Tarek Z Deeb, and Tim G Hales
August 1997, Canadian journal of microbiology,
Dai-Fei Wu, and Nidaa A Othman, and Douglas Sharp, and Arjun Mahendra, and Tarek Z Deeb, and Tim G Hales
June 2004, Biochemical Society transactions,
Dai-Fei Wu, and Nidaa A Othman, and Douglas Sharp, and Arjun Mahendra, and Tarek Z Deeb, and Tim G Hales
March 1998, Proceedings of the National Academy of Sciences of the United States of America,
Dai-Fei Wu, and Nidaa A Othman, and Douglas Sharp, and Arjun Mahendra, and Tarek Z Deeb, and Tim G Hales
February 1996, Molecular pharmacology,
Dai-Fei Wu, and Nidaa A Othman, and Douglas Sharp, and Arjun Mahendra, and Tarek Z Deeb, and Tim G Hales
October 2008, Biochimie,
Dai-Fei Wu, and Nidaa A Othman, and Douglas Sharp, and Arjun Mahendra, and Tarek Z Deeb, and Tim G Hales
September 2010, Plant physiology,
Dai-Fei Wu, and Nidaa A Othman, and Douglas Sharp, and Arjun Mahendra, and Tarek Z Deeb, and Tim G Hales
June 2002, Journal of molecular neuroscience : MN,
Dai-Fei Wu, and Nidaa A Othman, and Douglas Sharp, and Arjun Mahendra, and Tarek Z Deeb, and Tim G Hales
April 2006, Proteins,
Copied contents to your clipboard!